Oximacro®, a Cranberry Exract with the Highest Content of A-Type Proanthocyanidins (PAC-A) on the Market

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Transcript of Oximacro®, a Cranberry Exract with the Highest Content of A-Type Proanthocyanidins (PAC-A) on the Market

Oximacro®, a Cranberry Exract with the Highest Content of A-Type Proanthocyanidins (PACs-A)

on the MarketOximacro®, ProanthocyanidinsandUrinary Tract InfectionsE. coli interacts with receptors located in the epitelium and adheres to it.It has been estimated that 40% of women develop at least one UTI episode during their lifetimes (Micali et al. Crit Rev Food Sci Nutr. 2014;54:1063-1075).The berries of cranberry (Vaccinium macrocarpon Aiton) are used since hundreds of years as a remedy for diseases of the urinary tract and have attracted attention due to their potential health benefits.Guidelines on urinary tract infections of the European Association of Urology already include products made from cranberry among prophylactic methods of alternative UTI cure in women. Results suggest that presence of the A-type linkage in cranberry proanthocyanidins (PAC-A) may enhance both in vitro and urinary bacterial anti-adhesion activities and aid in maintaining urinary tract health (Howell et al. Phytochemistry. 2005;66:2281-2291).Administration of PAC-standardized cranberry powder at dosages containing 72 mg of Total PACs per day in two split doses of 36 mg in the morning and evening may offer protection against bacterial adhesion and virulence in the urinary tracts.(Howell et al. 2010, BMC Infect. Dis. 10, 94).However, the bioactivity of cranberry against UTI is dependent on the content of PAC-A (particularly dimers and trimers - Foo et al. 2000, J Nat Prod. 63:1225-1228).Take home message #1The more accurate is the PAC-A quantification, the more effective the cranberry extract will be.Chemical analysis of PACsCurrently, four methods are used to evaluate the content of cranberry PACs, two are based on depolimerization of PACs (e.g., the hydrochloric acid-butanol method known as Bates-Smith and the European Pharmacopoeia method), and two are colorimetric methods (a UV-VIS spectrophotometric method based on Prussian-blue or Folin-Ciocalteu reagents and the BL-DMAC method). The BL-DMAC colorimetric method (an aldehyde condensation of 4-dimethylaminocinnamaldehyde), appears to be more accurate than other methods and has been successfully used to quantify cranberry PACs (Prior et al. 2010, J Sci Food Agric. 90:1473-1478).BL-DMAC ASSAYBATES-SMITH ASSAY640 nm540 nmComparative study based on a standardized 5% PAC-A. Only BL-DMAC evaluates the real PAC concentration.The BL-DMAC Method does not distinguish PACs-A from PACs-BTiming of BL-DMAC analysis is important not to overestimate PAC-AComparative analysis between different concentrations of PAC-A and PAC-BTake home message #21) BL-DMAC is the best method for quantification2) BL-DMAC does not distinguish PAC-A and PAC-B3) BL-DMAC analysis timing is importantThe discrimination between PACs-A and PACs-B and their authentication is only possible by HPLC coupled to either fluorescence or mass detectors.Biosfered uses HPLC coupled to Ion Trap Mass Spectrometry to evaluate PACs quality and contentPACs A (black line) and PACs B (red line) are present in Oximacro®MS spectrum of PAC-A in Oximacro®~120 mg of Oximacro® => 36 mg PAC AThe highest PACs value, no matter the method used for quantification.Take home message #3Oximacro® offers the possibility to use a cranberry extract with the highest content of standardized and authenticated PACs-A, unprecedented on the world market.a new Cranberry extract with unique propertiesLeukocyte esterase (LE) is a urine test for the presence of white blood cells and other abnormalities associated with infection.The combination of the LE test with the urinary nitrite test provides an excellent screen for establishing the presence of a urinary tract infection (UTI)Take home message #7Oximacro® is also a potent antioxidantComparative AnalysisSTABILITYWe performed a series of tests in order to evaluate the response of Oximacro to temperature.Oximacro was tested for 180 days at room temperature (22 °C), at abused temperature (36 °C) and at stress temperature (55 °C). No significant (P=0.465; N=18) differences were observed between treatments in pH variations.Active water decreases with increasing temperature. A significant difference was found between 22 °C and 36 °C treatment (P=0.046; N=6); between 22 °C and 55 °C treatment (P=0.005; N=6) and between 36 °C and 55 °C treatment (P=0.018; N=6).No significant (P=0.129; N=18) differences were observed between treatments in PACs variations.Take home message #8Oximacro® is stable also at high temperaturePRODUCTIVITYTons/Year in our facility located in Turin, ItalyREFERENCE MATERIALVisit our web sitewww.biosfered.comAlso available as an alcohol-free fluid extract (36 mg PACs-A/ml)Antioxidant effect of Oximacro® with respecto to Trolox in vitro. No matter the test, Oximacro® shows a higher* antioxidant power.*The lowest the value the highest the activityAntioxidant ActivityClick on the left down corner of each video to start the movieOximacro® allows reduction from 800 mg or higher weight to 200 mg tablets, with evident advantages for customers.about 200%UrocultureBEFORE Oximacro®www.biosfered.comwww.biosfered.comwww.biosfered.comwww.biosfered.com

Urinary Tract Infections (UTIs) are widespread diseases that affect a large portion of the human population (about 13 million women each year in the United States and approximately 150 million people worldwide), with societal costs of approximately US$ 3.5 billion per year in the USA aloneSpectral analysis of BL-DMAC reaction of PAC-A (black line) and PAC-B (red line). When tested at the same concentration (20 µg/mL), PAC-B shows a higher absorbance at 640 nm with respect to PAC-ATime-course BL-DMAC reaction of 20 µg/mL PAC-A and PAC-B. When used at the same concentration, PAC-B (blue line) reacts faster and with a higher absorbance with respect to PAC-A (red line). Metric bars represent standard deviation.Prevention of Urinary Tract Infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study. Occhipinti et al. 2016, Urology Journal, 13: 2640-2649Oximacro® PACs content was assayed by the BL-DMAC method and the dimer and trimer PAC-A and PAC-B percentages were determined by HPLC-ESI-MS/MS. A balanced group of female volunteers ranging from 19 to over 51 years and males over 51 years was divided in two groups: The experimental group received 1 capsule containing Oximacro® (36 mg PAC-A) twice a day (morning and evening) for 7 days and the placebo group was given the same number of capsules with no PACs.Prevention of Urinary Tract Infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study. Occhipinti et al. 2016, Urology Journal, 13: 2640-2649Analysis of Oximacro® showed a high total PACs content (372.34 mg/g ±2.3) and a high percentage of PAC-A dimers and trimers (86.72% ±1.65). After 7 days of Oximacro® administration, a significant difference was found between placebo and Oximacro® groups for both females (Mann-Whitney = 875; P <0.001; N = 60) and males (Mann-Whitney = 24; P =0.016; N =10). Prevention of Urinary Tract Infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study. Occhipinti et al. 2016, Urology Journal, 13: 2640-2649Volunteers baseline characteristicsPrevention of Urinary Tract Infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study. Occhipinti et al. 2016, Urology Journal, 13: 2640-2649Boxplot representing the Ln scores of placebo and Oximacro® groups in both females and males volunteers. Females differences between placebo and Oximacro®: Mann-Whitney = 875; P <0.001; N = 60; males differences between placebo and Oximacro®: Mann-Whitney = 24; P =0.016; N = 10Prevention of Urinary Tract Infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study. Occhipinti et al. 2016, Urology Journal, 13: 2640-2649Boxplot representing the Ln scores of placebo and Oximacro® groups in both females (left panel) and males (right panel) volunteers according to the age range. Left panel, females age range 19/24, 25/30, 36/40, 41/50 and over 51 differences between placebo and Oximacro®: for each age range Mann-Whitney = 25; P =0.008; N = 10; females age range 31/35 differences between placebo and Oximacro®: Mann-Whitney = 20.5; P =0.095; N = 10. Right panel, males age range over 51 differences between placebo and Oximacro®: Mann-Whitney = 24; P =0.016; N = 10.Take home message #5Oximacro® used at 72 mg PAC-A is highly effective as a support for urinary tract health.Our pre-clinical study demonstrates that the use of dosages based on PAC-A instead of total PACs is strongly suggested.UrocultureBEFORE Oximacro®UrocultureAFTER 7 days Oximacro®UrocultureAFTER 7 days Oximacro®a new Cranberry extract with ANTIVIRALactivityInhibition of Herpes Simplex Type 1 and Type 2 infections by Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PACs-A). Terlizzi et al., Antiviral Research, 132 (2016) 154-164Take home message #6Overall, the described mechanism of action of Oximacro® against Herpes and Ifluenza Viruses advocates its potential application as a valid support for infections because of its high content of PAC-AInhibition of Herpes Simplex Type 1 and Type 2 infections by Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PACs-A). Terlizzi et al., Antiviral Research, 132 (2016) 154-164In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative to nucleoside analogues in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of its recognized anti-adhesive activity against bacterial pathogens, Oximacro® may represent a natural source of new antiviral microbicides.Inhibition of Herpes Simplex Type 1 and Type 2 infections by Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PACs-A). Terlizzi et al., Antiviral Research, 132 (2016) 154-164OXIMACRO® PACs-A ARE THE ONLY TO EXHERT ANTIVIRAL ACTIVITYIn order to evaluate whether the whole cranberry or the PAC-A only exerted a biological activity on Herpes virus we analysed the anti-HSV activity of the five Oximacro®-derived purified fractions. We found that only identified fractions 3 and 4, corresponding to dimers and trimers of PAC-A are responsible for the inhibitory activity of the whole extract (See table below)Antiviral activity of Oximacro® was tested in vitro on HSV-1 and HSV-2 replication. Pretreatment of Vero cells with Oximacro® 1 h before infection produced a significant concentration-dependent inhibition of both clinical isolates of HSV-1 and HSV-2 (see figure below)Analysis of the mode of action revealed that Oximacro® prevents adsorption of HSV-1 and HSV-2 to target cells. Prechilled Vero cells were treated with various concentrations of Oximacro®, or heparin at 4°C for 30 min and then infection was carried out with precooled HSV-1 or HSV-2 at a MOI of 0.002 for 3 h at 4°C in the presence of compounds as indicated in the figure below. Oximacro® impairs the attachment of HSV in a concentration-dependent manner and to a similar degree as observed in the virus yield.Therefore, Oximacro®, a cranberry extract highly enriched in A-type PACs, exerts a potent dose-dependent antiviral activity against clinical isolates of HSV-1 and HSV-2, the mechanism for which involves the inhibition of the initial virus attachment to the surface of target cells.The presence of A-type PACs in cranberry extract is central to its bioactivity. Gel filtration chromatography allowed us to fractionate Oximacro® into five major fractions, which were chemically characterized by HPLC-ESI-MS/MS and showed to contain anthocyanins, flavonoids, and PACs-A dimers and trimers(A) Eluogram from Sephadex-LH20 column fractionation of Oximacro®, five fractions were separated. The blue line correspond to absorbance readings at 360 nm, dotted orange line correspond to BL-DMAC reading at 640 nm. Only fractions 3 and 4 react to the BL-DMAC reagent.(B) HPLC-MS analysis of Fraction 1 was mainly composed of delphinidin and cyanidin glycosides and rutin. (C) HPLC-MS analysis of Fraction 2 shows the presence of quercetin and isorhamnetin.HPLC-MS analysis of Fractions 3 and 4 were dominated by several isomers of PAC-A dimers (D) and trimers (E), whereas fraction 5 did not contain any detectable compound.

THEREFORE, PACs-A ARE THE ONLY RESPONSIBLE FOR THE ANTIVIRAL ACTIVITY OF THE CRANBERRY EXTRACT OXIMACRO®. OTHER FRACTIONS PRESENT IN THE CRANBERRY EXTRACT DO NOT CONTRIBUTE TO THE BIOLOGICAL ACTIVITY AGAINST HSV-1 AND HSV-2.HOW DOES OXIMACRO® WORK AS AN ANTIVIRAL AGENT?The schematic representation shown in the next figure shows the interaction between the A-type PACs present in Oximacro® and the HSV envelope glycoproteins. Oximacro® binds the ectodomain of HSV glycoproteins (gD and gB) in a concentration- and time-dependent manner, thus inhibiting their functions in virus attachment and entry. However, at present, it remains still unclear whether the interactions between A-type PACs of Oximacro® and HSV envelope glycoproteins result in alterations of specific protein domains, or whether the A-type PACs simply “coat” the whole glycoproteins (as shown in the figure below), thus preventing access to their normal binding partners on target cells.See how the Bates-Smith and BL-DMAC methods workOximacro®: New Dietary Ingredient Notification report number 918

ANTIVIRALactivityagainstINFLUENZAThe cranberry extract Oximacro® exerts in vitro virucidal activity against influenza virus by interfering with hemagglutinin. Luganini et al., Frontiers in Microbiology, 9: 1826 (2018)The influenza viruses type A and type B (IAV, IBV) are widespread major pathogens among human populations and are responsible for seasonal epidemics and pandemics. Annual influenza epidemics cause worldwide 3–5 million cases of serious disease and up to half a million deaths among high-risk groups, with an even greater impact in developing countries, Oximacro® may represent a natural source of new antiviral microbicides.The cranberry extract Oximacro® exerts in vitro virucidal activity against influenza virus by interfering with hemagglutinin. Luganini et al., Frontiers in Microbiology, 9: 1826 (2018)Oximacro® inhibits both IAV and IBV replication in MDCK cells in a concentration-dependent manner. The calculated IC50 values were 4.5 ± 0.2 μg/ml for IAV, and 4.5 ± 0.5 μg/ml for IBV, respectively. The 50% cytotoxic concentration (CC50) was 141 ± 0.8 μg/ml, thus indicating that the antiviral activity of Oximacro® did not stem from a non-specific cytotoxicity. The selectivity index for IAV and IBV (CC50/IC50) was therefore 31.1 and 31.3, respectively (see figure below)Also against the influenza virus ONLY the PAC-A of Oximacro® have antiviral action. To pinpoint the active phytochemical of Oximacro® that was responsible for its inhibitory activity, antiviral assays were performed with purified fractions obtained through fractionation of Oximacro® (see above). Analysis of the fractions’ anti-IV activity identified fractions 3 and 4 as having inhibitory activity against IAV and IBV, thus indicating that the components responsible for the antiviral activity of Oximacro® were only PAC-A (See table below)THEREFORE, PACs-A ARE THE ONLY RESPONSIBLE FOR THE ANTIVIRAL ACTIVITY OF THE CRANBERRY EXTRACT OXIMACRO®. OTHER FRACTIONS PRESENT IN THE CRANBERRY EXTRACT DO NOT CONTRIBUTE TO THE BIOLOGICAL ACTIVITY AGAINST HSV-1 AND HSV-2.Oximacro® Targets Early Phases in the IV Replication CycleTo understand the Oximacro® mechanism of action, prechilled MDCK cells were infected with either IAV or IBV in the presence of different concentrations of Oximacro® for 2 h at 4°C, so that to enable the attachment of IV particles, but not their entry into target cells. Oximacro® inhibited the attachment of both IAV and IBV in a concentration-dependent manner (Figure A). Next, to investigate the inhibitory activity of Oximacro® on IV entry, prechilled MDCK monolayers were infected with IV at 4°C for 2 h to enable viral attachment but not entry. Different concentrations of Oximacro® were then added and infected cultures were incubated at 37°C for 2 h to allow entry of adsorbed IV particles. Oximacro® prevented the entry of both IAV and IBV in a concentration-dependent manner (Figure B). Together, these results revealed that Oximacro® is able to interfere with both viral attachment and entry, therefore suggesting its suitability as an early-acting inhibitor of IV replication.Therefore, Oximacro®, a cranberry extract highly enriched in A-type PACs, exerts a potent dose-dependent antiviral activity against clinical isolates of HSV-1 and HSV-2, the mechanism for which involves the inhibition of the initial virus attachment to the surface of target cells.Oximacro® Interferes With IV HemagglutininInfluenza virus hemagglutinin (HA) is responsible for both the IV attachment and entry by mediating both the initial interaction with cell receptors containing sialic acid, and the fusion between endosome membranes and the viral envelope. Oximacro® is able to interact with HA in a concentration- and time-dependent fashion and suggest that interactions between Oximacro® and IV HA may hamper its functions in virus attachment and entry, and underlie the overall antiviral activity of Oximacro®. To support the occurrence of interactions between HA and Oximacro® PAC-A further, in silico docking simulations were performed. Using the crystal structure of HA (A/Puerto Rico/8/1934, PDB ID: 1RU7) of our tested IAV strain, docking analysis revealed that PAC-A dimers binds to the internal grooves of the HA structure first (Figure A, left panel), and subsequently to the surface of the HA structure (Figure A, right panel). The best binding pose is shown in Figure B, where HA residues (PHE 299, TRP 234, and ASN 210) forming hydrogen bonds to PAC-A2 are highlighted.Therefore, Oximacro®, a cranberry extract highly enriched in A-type PACs, exerts a potent dose-dependent antiviral activity against clinical isolates of HSV-1 and HSV-2, the mechanism for which involves the inhibition of the initial virus attachment to the surface of target cells.